P10 – the Forgotten Baculovirus Hyperexpressed ProteinPublished on September 8, 2016
The ubiquitous polyhedrin gene promoter, from which most baculovirus expression vectors are developed, has an oft forgotten rival – the p10 gene promoter. The baculovirus expression system, on which our flashBAC™ vectors are based, derives from the original strategy whereby the virus polyhedrin gene coding region is replaced by the target recombinant gene sequences. These sequences remain under the control of the polyhedrin gene promoter to obtain high levels of protein production. Back in the 1980’s, this approach was chosen because removal of the polyhedrin gene sequences resulted in a virus-infected cell phenotype whereby production of polyhedra was abrogated. To say that these plaques could be readily distinguished might be overstating the case, but it was possible with patience to identify polyhedrin-negative plaques that could be tested subsequently for the presence of a foreign gene.
At about the same time that people were studying the polyhedrin gene, other groups were focused on the p10 gene. As its name implies, this gene encodes a protein of about 10 kilo Daltons, which is produced in the very late phase of baculovirus gene expression in virus-infected cells. In fact, it begins to be expressed slightly earlier than the polyhedrin gene, as we described in an earlier blog. It can also be deleted from the virus genome without having a deleterious effect on virus replication in cell culture. In fact, its removal can be beneficial for recombinant protein production as the virus-infected cells do not lyse in the latter stages on infection. This is one of the features of our flashBAC ULTRA variant, which can offer superior production of secreted or plasma membrane-targeted proteins.
The p10 gene is also expressed to levels comparable to polyhedrin. It might seem, therefore, to be an obvious vehicle to use as an expression vector. Unfortunately, unlike polyhedrin, it does not produce a readily identifiable phenotype in virus-infected cells. Observed under the light microscope, insect cells infected with a p10-negative genotype appear identical to those infected with the wild type virus. Although viruses lacking p10 do not cause cell lysis, this difference is sometimes not apparent until 5-6 days post infection, whereas polyhedrin-negative cells are obvious after 3 days. Pertinently, this effect on cell lysis was not noted until well after the baculovirus expression system was established using the polyhedrin gene promoter.
Expression systems based on the p10 gene locus were developed but these relied on removal of a copy of the bacterial beta-galactosidase coding region from the virus genome via homologous recombination to provide a blue/white selection. They have never caught on to rival the use of the polyhedrin gene.
However, where p10 has made a very significant contribution to baculovirus expression vectors is in the development of vectors for production of multiple proteins. Originally, baculovirus multi gene vectors relied on duplication of the polyhedrin gene promoter, but there were problems with genetic stability of the viruses derived. Fortunately, the p10 gene promoter can be duplicated in the virus genome upstream of the polyhedrin gene promoter to derive vectors (e.g. pOET5) for multiple gene expression. These expression cassettes can be duplicated so that several foreign genes can be inserted at the polyhedrin gene locus.
This does beg the question, “what about transcription terminators”? We will return to this topic in a future blog as we feel that it is a factor in expression that has been poorly described.